JPH02138299A - Immunoprotective antigen protein against pathogenic bacteria - Google Patents

Abstract

NEW MATERIAL: Protein which is recognized by a polyclonal anti-AC antibody which was generated to a specially purified adenylic acid cyclase(AC) and has no activity capable of adenylcyclase calmodulin (CaM)-activity, no affinity with CaM, about 43±4.3 K dalton of molecular weight Mr, and 5.7-6.1, especially 5.9 of isoelectric point Pi.

USE: Vaccine for pathogenic bacteria infectious disease.

PROCESS: For example, after Bordetella pertussis such as Bordetella pertussis 18323 (ATCC 9797) being cultivated, bacteria cells are collected and treated with the aqueous solution of urea, the urea extract is centrifuged, the supernatant is collected and treated with an anti-AC antibody to obtain immunity protective protein to pathogenic bacteria.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Pol-°--Persis, the causative agent of pertussis, indexes highly active adenylate cyclase, which has been shown to be the main toxin and immunoprotective antibody in experimental infections of mice. Secreted or released during proliferation.

This enzyme shares the property of being activated by a eukaryotic protein, namely calmodulin (CaM), with the secreted adenylate cyclase (edema factor) and eukaryotic adenylate cyclase of B. anthracis, Bacillus anthracis. do. Experiments with elevated AC preparations provide some evidence that this enzyme can enter various eukaryotic bacteria and subsequently promote a rapid but unregulated rise in intracellular cAMP. provided. This cAM
The P-mediated cytopathic effect is distinctly different from the deregulation of eukaryotic adenylate cyclase produced by other toxins, namely pertussis toxin.

It has been known since its discovery that B. peltasis undergoes a spontaneous phase change that is associated with a lack of expression of several phenotypic traits, including AC activity. However, these phenotypic changes are due to the expression (synthesis, transport and/or release) of molecules.
It is not clear whether this is due to either a lack of catalytic activity or a lack of catalytic activity.

By using anti-AC antibodies of one & [l, the inventors were able to detect A in toxic or non-toxic B peltasis mutants.
C antigen expression was examined and this immunological detection was correlated with enzyme activity.

By conducting these studies, we found proteins in toxic and non-toxic bacterial crude extracts that lack catalytic adenylate cyclase activity, but have beneficial antigenic properties.

It is an object of the present invention to provide in humans and animals novel proteins useful as immunoprotective antigens against Bordetella pertussis infection and the toxic effects of pathogenic bacteria.

The protein of the present invention has the following characteristics: - its molecular weight Mr is about 43 ± 4.3 K Daltons, - its isoelectric point Pi is 5.7 to 6.1, especially 5.9, - it is specifically recognized by a polyclonal anti-AC antibody raised against a purified active AC3Jl product, - that it lacks adenylate cyclase CaM activity, and - that it has an affinity for CaM. Lacking.

Molecular weight (molecular weight range Mr) was determined using ovalbumin as a control. The isoelectric point Pi corresponds to the electrophoretic migration when determined according to the method of O'Farrell et al. in reference (6), which is given at the end of this specification.

Such a protein, which is inactive with respect to catalytic adenylate cyclase oxygen activity, is structurally antigenically similar to the active AC of Bordetella pertussis, and more particularly -1-:j4''-2ino. is noteworthy.

According to the invention, it has been found that inactive and active adenylate cyclases have at least a common epitope.

The terms "inactive" and "active" as used herein refer to the enzymatic activity of adenylate cyclase.

The protein is also present in a high molecular weight form of about 90 Daltons and has the same properties as the protein, in particular the same P
i and suggests that it is a dimer.

The proteins of the invention can be obtained from virulent bacteria and mutants thereof expressing active AC, more particularly from pathogenic bacteria, which AC is capable of interfering with the AC of eukaryotic cells. Therefore, both active and inactive forms are present in the bacteria. Only the active form is secreted. However, Dan, El? The 9-person culture contains only the inactive form.

The protein can be obtained from Bordetella pertussis. Bordetella pertussis used to obtain the proteins of the invention is a bacterium that is responsible for respiratory diseases in vertebrates,
For example, in humans, there are ±kj- and Menggo'yumi and'JyyotaLpo, Pera: Shikairinosu, and in animals, there are Kodo Emenritsu Broncaptica and Mesoi Avium. .

Cross-reactive proteins having a different molecular weight than one of said proteins can also be characterized from the ffi system, Yersiniae and Passeurae.

Said antigenic proteins and cross-reactive antigens can also be obtained from non-toxic variants of adenylate cyclase whose oxygen activity has not been previously found.

Variants of said virulent and non-virulent strains can be used according to the invention as they produce cross-reactive antigenic molecules.

Bacterial extracts which are advantageous for the extraction of cross-reactive proteins and molecules according to the invention can be obtained, for example, by treating bacterial cells expressing AC with urea and collecting the supernatant after centrifugation. obtained by doing. Any other type of bacterial lysis or molecular cloning can be used as a source for the protein.

The proteins of the present invention include dan-2 υi otiri and dan.

The protein can be purified through immunoaffinity with a specific antibody against the native adenylate cyclase of PJ9.

The study of mouse models of infection conferred by bacteria synthesizing the proteins of the invention has made it possible to point out their antigenic properties.

It appears that said protein is indeed capable of inducing protection. This is because certain antibodies or B
This is because it can passively provide sufficient protection against intranasal challenge by barbell cis.

Toxicological assays are performed on the bacteria producing these antigens, which show the absence of toxicity when administered intranasally or parenterally to mice, and the purified proteins are tested to determine whether they are processed or not. It can be conveniently used for the preparation of vaccine main components, provided that it does not show cross-reactivity with the eukaryotic proteins that have been used.

The vaccines of the invention are characterized in that they are molecular vaccines comprising said antigenic proteins together with a pharmaceutical carrier.

Conventional dosages and pharmaceutical forms are used.

In the vaccine compositions of the invention, the antigenic protein may or may not be combined with PHA in the same inoculum.FHA may be administered at the same time as adenylate cyclase or at different times. Dew. The FHA preparation is
For example, Sa to etc., Infecf.

I m5un, 19B3, 41, pages 313-320 or I maizumi et al., Journal of
Microbiol, Methods 2゜33
4-347 (1984).

In another embodiment, the antigenic protein is 198
FR Patent Application No. 86159 filed on November 17, 2016
63, and optionally also with PHA.

Advantageously, the vaccine composition of the invention is particularly suitable for
prevent infections caused by i.

Vaccination is conveniently carried out by intranasal, oral or parenteral routes.

Therefore, active immunization against intranasal infection with K-di-4m is obtained with a whole cell vaccine prepared from non-toxic AC Dan-25-Yin-Niru.

The invention also relates to polyclonal antibodies directed against said proteins as novel products.

These antibodies are obtained by immunizing animals, such as rabbits or mice, with the protein in question and by collecting the antisera containing said antibodies and the antibodies themselves by standard methods.

The invention also relates to monoclonal antibodies obtained by fusion of mouse myeloma with lung lymphocytes from mice immunized with the purified protein.

These monoclonal immunoglobulin preparations are characterized in that they specifically recognize epitopes of purified proteins leading to the induction of the synthesis of protective antibodies.

Advantageously, the antibodies of the invention exert an immunoprotective effect against the pathogenic effects of said bacteria.

Other features and advantages of the invention will be given in the following examples. The materials and methods used in this example are as follows. : Box 11 and then 51>X18323 (ATCC9
797) or the AC-mutant of Borde
+ -Gen-gou from cultures on blood agar plates (1).

Calmodulin-affinity purification of AC from periplasmic and extracellular extracts, radioimmunoassay (RIA) and quantification of enzyme activity were performed as described in (2), (3) and (4), respectively.

Control RIA was performed using bovine serum albumin (BSA) to check for nonspecific inhibition by concentrated protein preparations.
) was assayed with a concentrated solution of

Mouse polyclonal and monoclonal antibodies raised against the affinity calmodulin purified molecule (2
). Wangketsu Sakosei etc. 1 standing l five bed orbit and 5O5-
PAGE electrophoresis was performed according to O' Farrell (6). Ampholine pH3~10 (P
ha-rmacia) and pH 4-9 (5erval
yte) was used.

Protein concentrations were determined using BSA as a control.
Determined as conventionally by Nafder's method (7).

After 5OS-PAGE, proteins were transferred to nitrocellulose by the Towbin method (8). After transfer, the nitrocellulose was treated with 50 mH Tris H (l (pH 8), 15
It was saturated with 5% milk powder in 0 mM NaCl solution (buffer A) and incubated with mouse antibodies diluted in milk buffer. After washing with buffer A, the two exposures were performed with 3,3-diaminobenzidine (
500g/ml), 0.03%Hz (h+100m
M's TrisHcJ! Horseradish peroxidase-conjugated goat anti-mouse Ig (Biosys France) or phosphatase-conjugated antibody (Biosys F r
ance).

1. Immunoplot of bacterial periplasmic extracts with anti-AC antibodies.

By various anti-AC antibodies. Immunoplots of bacterial periplasmic extracts from toxic or non-toxic Dan-Nito Takanohisa (5OS-PAG with 7.5% acrylamide)
E) was performed.

The extracts were urea extracts of -W-254''-2, AC'' of 1832B and AC-mutant obtained by (3).

They were reacted with: A/polyclonal antibodies raised against the purified AC preparation and B/monoclonal antibodies raised against said purified AC preparation.

Those purified ACgJl products are those disclosed in the FR patent application (reference 2) in the name of the applicant.

Such preparations ensure that they have a high degree of purity and are free from contaminating bacterial substances, in particular pertussis toxin, lipopolysaccharide (or
PS) and fibrillary hemagglutinin (or FHA). Therefore, AC can be obtained in homogeneous form, precipitating with an S factor equal to 3.6 in a sucrose density gradient and existing in two structurally related molecular forms of 45 and 43 K Daltons, respectively.

According to another point of view, ACgll products are capable of achieving and even more than 1600 μmol of c
It is characterized by having an activity of more than 0 mg AMP/min.

Said purified AC preparation can be obtained by contacting calmodulin with previously concentrated supernatants of bacterial cultures exhibiting adenylate cyclase or extracts of these bacteria.

Polyclonal antibodies are produced by immunizing an animal, such as a rabbit or a mouse, with the ACI preparation in question, in which AC is present in free form or in the form of a complex with a eukaryotic protein, in particular calmodulin, and then producing an antiserum containing the antibody. and then the antibody itself is produced by recovering it by standard methods.

Monoclonal antibodies can be obtained by fusion of mouse myeloma with lung lymphocytes from mice immunized with purified adenylate cyclase.

Monoclonal antibodies (8-25) are particularly used. They immunoprecipitate triplets of 50, 45, and 43 K Daltons and proteins with molecular weights above 100 Daltons in crude extracts and preparations of enzymes purified from bacterial cells.

The hybridoma strain that produces monoclonal antibodies is 1
October 9, 986, Nation-n as l-610
al Co11ection of Culture
s of Microorga-nisss (CNCM
) was deposited at

The immunoplots obtained are reported in Figure 1, where in par) A and B corresponding to the above reactions, lane l
and 3 correspond to reactions with crude extracts of AC'' and AC-mutants, respectively, and lanes 2 and 4 correspond to the same extracts after depletion of calmodulin-binding proteins.Activity of (1) is 2.000 units, the activity of (2) is 100 units, and the activity of (3) and (4) is 1 unit or less.

FIG. 1 shows that the extract exhibits different patterns of recognition by different antibodies.

In the bacterial urea extract (lane 1), there are three bands with Dalton at Mr 43, 45 and 90, respectively, and a pure homogeneous AC with Dalton at 43-45.
This was seen in the Western plot with polyclonal antibodies raised against the complex. After depletion of calmodulin-binding molecules from crude AC'' urea extract, 45
Dalton's band disappeared in .

Western plot analysis of AC'' urea extracts with a monoclonal anti-AC antibody showed that only calmodulin-binding molecules were recognized by this antibody.

This antigen can be polyclonal or monoclonal anti-A
C antibody was not detected in the AC-crude urea extract (lanes 2 and 4).

Activity quantification of these various extracts showed that the disappearance of Dalton's band at 45 correlated with a significant loss of AC activity. In the urea extract, the AC" activity is about 1,000 to 2,500 units (one unit is 30°C
, 1 nanomole of cAM formed per minute at pH 8
), and this crude extract corresponds to CaM-
There were only 20-40 units after passing through the affinity column. CaM-affinity eluting molecules are recognized by poly or monoclonal antibodies and are detected at 8,000 per m42.
It had a cyclase activity of ˜20.000 units and an apparent Mr of Dalton of 45.

2. Immunoblot on a flat polyacrylamide gel: Perform immunoplot on a flat polyacrylamide gel.
The gel contained: ■) CaM purified AC expressed with a monoclonal antibody (the same image is obtained with a polyclonal antibody), pi = 5.
1, Dalton at Mr = 45, labeled rabbit IgG anti-mouse peroxidase, diethyl-amino-benzidine (DAB): ■) Crude urea extract of the virulent variant (same as in Fig. 1).

The resulting immunoplots are 2I and 2I, respectively.
As shown in the figure. In the above figure, A and B correspond to the following monoclonal antibodies, IgG peroxidase, DA
First detection with B (A) Second detection with one labeled polyclonal antibody anti-mouse phosphatase (crude extract represented only by polyclonal antibody shows the same picture as A+B).

Purified AC was detected by both monoclonal and polyclonal antibodies (Figure 21), and it
Dalton on r45 and isoelectric point (pi) of 5.2-5.1
It had This pi value was confirmed from I2sl labeling of CaM affinity purified AC detected by autoradiodram.

AC" urea extract (Figure 2) shows three molecules with polyclonal antibodies and their Mr and pi
In , one is 45 to Dalton and 5.2 to 5.1
have a pi of 43K Dalton and 90K respectively.
*Px5-5 with Dalton and pi of 6-5.8
．． These two bands at 8 were recognized during the flow after loading onto the CaM affinity column.

The monoclonal antibody was 4
5 showed a band only in Dalton, and after the AC" urea extract was passed through the CaM affinity column, it no longer showed any bands.

+9) Incubate the polyclonal anti-AC antibody with a strip of nitrocellulose on which the antigen has already been transferred by Western Blot, and incubate the strip so that it simply has an antigen preparation limited to proteins of Mr 43K Dalton. was severed. +1 sized strips were selected from those previously immunoplotted on control sample pieces of individual Western plots representing 43K Dalton antigen with anti-AC polyclonal antibodies.

Elution of antibodies immobilized on nitrocellulose strips was performed by P
This was achieved by H2,8 H(J) buffer followed by immediate neutralization with phosphate buffer. These antibodies were tested for specificity against various antigen preparations.

These antibodies, which react with molecules homologous to Dalton in C.
aM-affinity purified 45 cross-reacted with Dalton's AC.

In a radioimmunoassay, inhibition of immobilization of 1″% I-labeled CaM-bound AC onto a polyclonal antibody was assayed by l-Odoshanohisa extract. As shown, purified AC and urea extracts of AC'' bacteria and culture supernatants blocked immobilization in a similar manner.

AC-supernatant fluid or urea extract is anti-AC against AC.
Antibody immobilization was weakly inhibited.

The % inhibition by various bacterial extracts varied as a function of the relative protein concentration of the samples.

Heat-inactivated vaccine (80°C) administered subcutaneously in a single infection per immunization provided a protection rate of 50%.

The documents mentioned in this specification are shown below: (1) B
r+'zin, C, etc., +FBMS Micr
obiol.

Lett, 42. pages 75-80, 1987゜(2
) I n5titut Pa5teur and
French Patent Application No. 8615963 filed on November 17, 1986 in the name of In5erts.

(3) L adant, D, etc., J,
B iol, Che+m, 261°16264~1
6269 pages, 1986゜(4) White A
, A, etc., ,Methods Enz mol.

38G, pages 41-46, 1974. For example, Ha
Method improved by nou-ne etc. + J, B
iol, Chem.

252, pages 2039-2046, 1977゜(5
) Lae++v+Ii Nature, Lon
clres+ 227. pages 680-685,
1976゜(6) O' Farrell, J Bi
ol, Chen+,, 1975°250, 4007
~4021 pages.

(7) 5chnafter, W, etc., + Ana
l, B 1och-+1973, 56.502-51
4 pages.

(8) Towbin, H. (1979), Pr.
oc, Natl, Acad.

Danhong, Lihong 76, 4350 pages, and (9) N
ghiem H, 0, 1988+ Journal
Of Imm! Jnolo 1cal Methods
, published by JIM 04806.

[Brief explanation of the drawing]

Figure 1 represents the immunoplots of extracts from toxic or non-toxic l-tamadori with various anti-AC antibodies; FIG. 3 depicts immunoplots of planar gel electrophoresis of AC or extracts, and FIG. 3 depicts radioimmunoassay. These drawings are photographs and are photographs in place of drawings. Engraving of drawings (no change in content) 94 to 67 - 43K □ --- Nawateha, FIG, 2 II 494 to 467, 443K H FIG, 21 FIG, 3 5.9 Procedural amendment (method) % Formula % Display of the case 1999 Patent Application No. 96539 Name of the invention Person who corrects immunoprotective antigen protein against pathogenic bacteria Relationship to the case Patent applicant name Instituille Basteur (1 stone outside) 4. Agent address 〒 105 Toranomon-chome-8-10-6, Minato-ku, Tokyo, subject of amendment (1) “Representative of applicant” column of application (2) Power of attorney (3) Drawing 7, contents of amendment 11) (21 As attached (3) Engraving of drawings (no change in content) 8, list of attached documents (1) Application for correction (2) Power of attorney and translation (3) Engraving drawings (4) and $ 1 1 copy 1 each 1 1 prefecture

Claims (1)

Claims: 1. Recognized by polyclonal anti-AC antibodies specifically raised against purified adenylyl cyclase (AC) preparations, lacking adenylyl cyclase calmodulin (CaM)-active activity; and a protein lacking affinity for CaM, having a molecular weight Mr of about 43±4.3 K Daltons and an isoelectric point Pi of 5.7-6.1, especially 5.9. 2. The protein of claim 1 having a high molecular weight of about 90K Daltons. 3. Toxic or non-toxic Bordetella pertasis (Bord
etella pertussis), Bordetella parapertans
ussis), Bacillus antrans
s antoracis), Yersiniae (Yersi
niae) and Pasteurellae (Pasteurell)
3. A protein having antigenic cross-reactivity with the protein according to claim 1 or 2 (but having a different molecular weight), which can be obtained from Ae) as well as variants thereof. 4. Bordetella pertasis, Bordetella parapertans, Bordetella bronchiseptica (Bord
etella bronchiseptica) and Bordetella avium
) and their variants. 5. Preparation of polyclonal antibodies formed against the purified protein according to any one of claims 1 to 4. 6. A monoclonal antibody obtained by fusion of mouse myeloma with lung lymphocytes of a mouse immunized with the purified protein according to any one of claims 1 to 4. 7. Use of the antibody according to claim 5 or 6 as a protective substance. 8. A vaccine according to any one of claims 1 to 4, characterized in that it is a molecular vaccine, capable of inducing protection against infection and toxic effects by pathogenic bacteria in pharmaceutical carriers, humans and animals. Vaccine which may or may not contain in combination the mentioned proteins and optionally FHA (filamentous hemagglutinin) and/or manufactured AC in the same inoculum. 9. Vaccine according to claim 8, characterized in that it is present in the form of an intranasal, oral or parenteral preparation.